The amyloid -protein (A), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is formed by the sequential cleavage of the amyloid -protein precursor (APP) by - and ?-secretases. ?-Secretases cleaves the APP transmembrane domain at the ? site, producing the C-terminus of A, and at the ? site, producing the N-terminus of the APP intracellular domain. The goal of this project is to understand the process by which the ?--secretase complex trims long A peptide intermediates into shorter forms and how disease- causing PS mutations alter activity. With these goals in mind, the following specific questions will be addressed: (1) Which shorter A peptides are produced via the trimming of specific long A peptides? We will identify and quantify all A peptides formed from normal ? cleavage products A49 and A48. We will also determine if A50, which is not a ? normal cleavage product, leads to the formation of the otherwise unobserved A47, A44 and A41. The result will help answer the question of whether trimming by every 3 residues is a general rule, and if so, how rigid is this rule. (2) How does ?-secretase accomplish C-terminal tripeptide trimming of long A peptide intermediates? Evidence supports initial ? proteolysis to produce long A peptides and then cleavage every 3 residues, but the mechanism by which this occurs is unknown. We hypothesize that the newly formed carboxy-terminus of long A produced upon ? cleavage by ?-secretase is critical for trimming and with apparent precision by every 3 residues. To test this hypothesis, we will examine the ability of C-terminal amides of long A peptides to serve as substrates. (3) What are the effects of Alzheimer-causing PS1 mutations on the trimming of long A peptides? We have shown that such PS1-mutant ?-secretases complexes can increase the proportion of long-to-short A peptides from recombinant APP substrate. Here, we will test the conversion of A49 and A48 by these mutant protease complexes, examining the A products that are formed, the proportion of these products, and their rates of formation relative to the wild-type complex. We hypothesize that the disease-causing PS1 mutations increase the proportion of long A peptides by slowing down the trimming process in general. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand ?-secretase, a complex enzyme critical to the cause of Alzheimer's disease. Biochemical experiments are proposed to test hypotheses about how this enzyme works and how it is dysregulated in Alzheimer's disease.